The present application relates to nucleic acid isothermal amplification methods, specifically to nucleic acid isothermal amplification methods in which the reverse transcription reaction of the target RNA strand into the template DNA strand is performed in a series of procedures with the amplification reaction of the template DNA strand.
PCR (Polymerase Chain Reaction) has been used as a method of nucleic acid amplification method. In PCR, template DNA strands are amplified by the repeated cycles of three temperature steps including (1) heat denature, (2) annealing, and (3) extension reaction.
In the first heat denature step, the template DNA strands are dissociated into single strands from the double-strands. The reaction temperature for the heat denature is generally about 94° C. In the second annealing step, oligonucleotide primers are bound (annealed) to the single-stranded template DNA strands. The annealing reaction temperature is generally about 50° C. to 60° C. In the third extension reaction, DNA polymerase synthesizes a DNA complementary to the single-stranded portion, using the oligonucleotide primer site as the origin. The reaction temperature for the extension reaction is generally about 72° C.
For gene expression level analyses and cDNA cloning, RT-PCR (Reverse Transcription-Polymerase Chain Reaction) is used in which the reaction of the reverse-transcription of mRNA into cDNA is performed preceding PCR. In RT-PCR, a single-step RT-PCR method has widely been used in which the preceding reverse transcription reaction and the next amplification reaction are performed in a series of procedures.
In the single-step RT-PCR method, a DNA strand (cDNA) complementary to the RNA strand (mRNA) under expression analysis or being cloned is first synthesized by reverse transcription reaction. The reaction is performed with a reaction solution that contains the RNA strand, reverse transcriptase, and reverse-transcription oligonucleotide primers, maintained at a temperature of generally about 42° C. In the next amplification reaction, a PCR reaction is performed using the synthesized DNA strand as a template. The primers used for the amplification reaction are generally selected to bind to the RNA strand (or template DNA strand) base sequence at sites different from the binding sites for the oligonucleotide primers used for the reverse-transcription.
In recent years, an easier method, called isothermal amplification, has come to be used as a nucleic acid amplification method that can obviate the need for the repeated temperature cycles. For example, in LAMP (Loop-Mediated Isothermal Amplification), template nucleic acid strands are mixed with reagents such as oligonucleotide primers, strand displacement-type DNA synthetase, and nucleic acid monomer, and the mixture is held at a constant temperature (in the vicinity of 65° C.) to run the reaction.
In connection with the present disclosure, Light-Triggered Polymerase Chain Reaction, Chem. Commun., 2 008, 462-464 describes a technique for controlling PCR reaction with the use of an oligonucleotide primer that includes a photodegradable protecting group-attached thymine in its base sequence. In this technique, a 6-nitropiperonyloxymethyl (NPOM) group that dissociates by irradiation of UV rays is used as the photodegradable protecting group.